During cardiac development, valvular progenitors develop from cardiac endothelium in response to a TGFbeta-mediated tissue interaction between the myocardium and the endothelium. This "epithelial- mesenchymal cell transformation" of endothelial cells has been alternatively postulated to be due to the regulation of a "master gene", the result of changes in ECM or its receptors, or the loss of cell-cell adhesion by endothelial cells. While aspects of each of these hypotheses are likely to be involved, adhesion by endothelial cells. While aspects of each of these hypotheses are likely to be involved, data obtained in this laboratory suggest that a complex regulatory process takes place during cell transformation. It is our hypothesis that epithelial-mesenchymal cell transformation is the result of coordinate regulation of several transcription factors by different signal transduction pathways. To explore this hypothesize we will focus on the regulation of two critical events. The first is an activation process characterized by a change in cell morphology and the second is a process is an activation process characterized by a change in cell morphology and the second is a process wherein cells invade the underlying ECM. These events appear to correlate with two specific transcription factors, Slug and Mox-1. Slug functions early in the cell activation process while Mox-1 functions during cellular invasion. A third transcription factor found in the heart, Snail, will be investigated as it may have a redundant activity with Slug. We previously showed that several distinct transduction pathways mediate endothelial responses to the transforming stimulus. The next phase of the work is to link signal transduction with specific gene regulation. We will use a variety of reagents to determine whether Mox-1 and Slug are regulated in parallel or in serial by the inductive stimulus. Specific aims are: 1. Extend characterizing of Mox-1 and Slug regulation in endothelial and mesenchymal cells by various signal pathways. 2. Identify promoter/enhancer elements in Mox1 and Slug upstream sequences that mediate responses to specific signal transduction pathways. 3. Test involvement of Smads in the regulation of Slug, or Mox1 via a TGFbeta the time of cell transformation. Together, these aims will further the characterization of the fundamental process of cell development in the heart.